PROJECT SUMMARY Only about dozen mass spectrometry (MS) protein tests have been translated into clinical laboratories since the MALDI and ESI approaches were developed thirty years ago. While the cost and complexity of these tests are important factors impeding their clinical adoption, new content generated via proteoforms detection could provide the impetus for further development and translation. Our recent studies have revealed strong associations between relative abundance of a specific apolipoprotein C-III proteoforms and plasma triglycerides concentrations in multiple cross-sectional and longitudinal cohorts, and also suggested prognostic and possible mechanistic role in dyslipidemia and cardiovascular disease risk. These intriguing results warrant further large-scale investigations to verify the biomarker potential of these proteoforms in lipid metabolism and cardiometabolic risk. We propose to develop and demonstrate a low-cost, high-throughput MALDI-TOF MS apoC-III proteoforms test that will find immediate use in clinical utility studies of these proteoforms. The test workflow will include immunoaffinity isolaton of apoC-III from minimal amounts of human plasma with antibody-coated 96-well plates, and elution onto MALDI targets for MS analyses and determination of the proteforms relative abundance. The steps of antibody immobilization, sample dilution, assaying, rinses, and elution onto MALDI targets will be optimized. The number of assay steps will be kept to a minimum, to keep the cost per test low. Reproducibility of the new test will also be evaluated. The proposed MS test is innovative in its simplicity, cost, and content delivered - it will transform clinical research and diagnostics by enabling fast and cost-effective screening of apoC-III proteoforms. Similar tests for other proteins can also be developed, significantly expanding our ability to study human proteoforms and their role in disease diagnosis, therapy monitoring, and outcome.